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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 1  |  Page : 127-131

Differential diagnosis of non-small cell lung carcinoma by circulating microRNA


1 Department of Biochemistry, AIIMS, Rishikesh, India
2 Center For Advance Research, Stem Cell/Cell Culture Lab, King George's Medical University, Lucknow, Uttar Pradesh, India
3 Department of Pulmonary Critical Care, King George's Medical University, Lucknow, Uttar Pradesh, India

Date of Submission16-Oct-2019
Date of Acceptance07-Jan-2020
Date of Web Publication29-Apr-2020

Correspondence Address:
Satyendra Kumar Singh
Center For Advance Research, Stem Cell/Cell Culture Lab, King George's Medical University, Lucknow, Uttar Pradesh
India
Ved Prakash
Department of Pulmonary Critical Care, King George's Medical University, Lucknow 220 003, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_872_19

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 > Abstract 


Introduction: More than 70% of lung cancer comprises nonsmall-cell lung carcinoma and is associated with poor survival outcome owing to late diagnosis. Identification of lung cancer in early stages when no clinical signs or symptoms are evident, can drastically improve the prognosis. To this end, we aimed to evaluate the changes occurring at tissue level by assessing the expression of six microRNAs (miRNAs) in lung adenocarcinoma (AC) and squamous cell carcinoma (SCC).
Materials and Methods: Peripheral blood of histopathologically proven cases of lung AC and SCC was collected and processed for the isolation of miRNAs using commercially available kit. Primers against mir-2114, mir-2115, mir-2116, mir-2117, mir-449c, and mir-548q with loading control Caenorhabditis elegans were used. Screening was carried out in thirty cases of both AC and SCC, whereas twenty healthy controls were included.
Results:Real-time polymerase chain reaction data revealed that the expression of mir-2114 and mir-449c in AC and mir-2115 in SCC was significantly upregulated. The expression of these miRNAs was also confirmed in lung AC cell line. The differential pattern of expression of these miRNAs can be used for precise diagnosis of lung carcinoma
Conclusions: We have used a noninvasive technique to identify the subtype of lung cancer based on molecular genetic signatures. The results suggest that through molecular profiling of miRNA, we can screen high-risk cases for cancer interception.

Keywords: MicroRNA, molecular diagnosis, nonsmall-cell lung cancer, real-time polymerase chain reaction


How to cite this article:
Singh A, Kant R, Saluja TS, Tripathi T, Srivastava K, Naithani M, Gupta A, Mirza AA, Prakash V, Singh SK. Differential diagnosis of non-small cell lung carcinoma by circulating microRNA. J Can Res Ther 2020;16:127-31

How to cite this URL:
Singh A, Kant R, Saluja TS, Tripathi T, Srivastava K, Naithani M, Gupta A, Mirza AA, Prakash V, Singh SK. Differential diagnosis of non-small cell lung carcinoma by circulating microRNA. J Can Res Ther [serial online] 2020 [cited 2020 Jun 6];16:127-31. Available from: http://www.cancerjournal.net/text.asp?2020/16/1/127/283319




 > Introduction Top


On global scale, lung cancer remains the leading cause of cancer-related deaths over the past few decades. According to Global cancer statistics 2018 (GLOBOCAN), lung carcinoma was the most commonly diagnosed malignancy with an incidence rate of around 12% of total cancer cases. It was the main cause of cancer-related deaths in males, and its mortality rate was found to be the highest among all cancer cases.[1] This high mortality is generally due to late diagnosis which reduces the available treatment options, and thereby, effective management becomes difficult.

The classical diagnostic tools for lung cancer detection include chest X-ray, computed tomography (CT) scan, positron-emission tomography (PET) scan, magnetic resonance imaging (MRI), and ultrasound. Chest X-ray is effective in detecting tumors but has no concomitant effect on the survival rate of lung cancer patients. CT and PET scans have improved efficacy in disease diagnosis and treatment but have no significant survival benefit. These diagnostic techniques are mainly based on tumor morphology and appearance and can only be used when the tumor formation has already initiated. This limitation of classical diagnostic techniques has made them incompetent in the early detection and prevention of lung cancer.[2],[3]

Conventional diagnostic therapies, being inefficacious, set up the need for the development of cutting-edge technologies for early detection. In this context, molecular diagnostics emerged as the most potent and transformative area of diagnosis. It is a subset of diagnostic techniques that evaluates diseases causing changes at the molecular level. Most of the bodily processes are carried out by the interplay between our genes and the proteins. Any dysregulation in these interactions can disrupt normal cellular functioning and initiate tumor formation.[4] Hence, if the detection is made at the level of molecular biomarkers, there is a chance of improved clinical outcomes.

The advancements in molecular techniques have facilitated the use of these molecular biomarkers in the clinical setup. Present-day techniques such as polymerase chain reaction (PCR), nucleic acid hybridization, Sanger sequencing measure the level of these biomarkers and allocate its expression to a particular state of body. In this way, the expression profiling of biomarkers can be used for the identification of lung cancer as well as for determining its stage. The use of microRNAs (miRNAs) as biomarkers for the diagnosis of cancer cases started with characterization of disease-related miRNA in lymphocytic leukemia. miRNAs are endogenous, single stranded, small stretch (~22 nucleotides) of nucleic acids which are noncoding but have regulatory functions. They bring about the post translational modification of gene expression by binding to their target mRNA and inhibit their degradation. This small class of RNA has a significant role in a variety of metabolic processes such as differentiation, apoptosis, cell development, and proliferation. Owing to their regulatory effect on gene expression, miRNAs are potential biomarkers for early detection of different pathophysiological conditions. A significant number of miRNAs are found extracellularly and are remarkably stable in bodily fluids. Estimating their levels in circulation offers several opportunities for developing blood-based biomarkers in cancer diagnostics.

miRNAs are frequently deregulated in cancer and their expression is usually tissue specific. Deranged levels of miRNA have been linked to several disease processes such as drug-related liver injury (miR-122), myocardial infarction (miR-499), and prostate carcinoma (miR-141).[5],[6],[7] In this study, we aimed to characterize the expression of five miRNAs in blood sample of histopathologically diagnosed case of lung adenocarcinoma (AC) and squamous cell carcinoma (SCC). We also ascertained the level of these miRNAs in AC and SCC for molecular differential diagnosis in lung cancer. Our results lay the foundation for early diagnosis of lung cancer using blood-based biomarkers and raise the possibility of tracking aberrant cellular changes after treatment with blood levels of miRNA for an effective patient management.


 > Materials and Methods Top


Clinical sample and microRNA isolation

The study was approved by the institutional ethical committee. Newly diagnosed and histopathologically proven cases of lung AC and SCC were included in this study. Cases with previous history of any malignancy and treatment or with a history of any other systemic disease were excluded from the study. Peripheral blood samples were obtained from histopathologically diagnosed thirty AC and thirty SCC cases, whereas twenty control samples were obtained from healthy individuals. The blood samples of lung cancer patients were collected after getting written consent from the donor or guardian.

The samples were collected in ethylenediaminetetraacetic acid vacutainer to prevent clotting. The plasma fraction for isolation of miRNA was prepared according to the standard protocol. The blood was transferred to sterile tubes and centrifuged at 1100 × g for 12 min at 4°C to separate blood cells and plasma. Thereupon, miRNA was isolated from 200 μl of plasma using Qiagen miRNA easy serum/plasma Kit (Qiagen, USA) as per manufacturer's instructions. Briefly, Trizol reagent (Qiagen, USA) was added to the plasma samples, followed by vortexing to allow proper mixing. After incubation for 5 min at room temperature, miRNeasy serum/plasma spike-in-control was added. An equal volume of chloroform was added for phase separation, and the sample was centrifuged at 10,000 × g for 15 min at 4°C. Aqueous phase obtained after phase separation was transferred to a fresh collection tube, and ethanol was added. The sample was transferred to RNeasy MinElute spin column and centrifuged for 15 s at 10,000 g. The column was again washed with standard buffers to remove salts and impurities. Following incubation with RNase free DNase for 5 min at room temperature washing was carried out with buffers, and miRNA was eluted with DNase RNase free water. The concentration of miRNA was estimated using NanoDrop™ (Thermo scientific™, USA). cDNA was synthesized with 200 ng total miRNA with HiSpec buffer using miScript II real-time (RT) Kit (Qiagen, USA) according to manufacturer's protocol.

Real-time reverse transcription-polymerase chain reaction

Commercially available primers for miRNA 2117, miRNA 548q, miRNA 449c, miRNA 2115, miRNA 2116, and miRNA 2114(Accession No-MI0010633, MI0010637, MI0003823, MI0010634, MI0010635, and MI0010636, respectively) were used. Caenorhabditis elegans (Ce miR-39_1) was used as loading control (miScript primer assay, Qiagen, USA).

RT-PCR was carried out using SYBR green reagent (Qiagen, USA). Samples were run in triplicate in a 96 well plate (ABI, Thermo Scientific, USA) with 2 ng of cDNA on STEPONE PLUS™ RT-PCR. The RT-PCR run program consisted of a holding stage of 95°C for 10 min, followed by 45 cycles of 15 s at 95°C, 30 s at 60°C 1 min at 70°C, and 1 h of melt curve stage.

Real-time-polymerase chain reaction analysis and statistics

miRNA expression levels were measured by miRNA mini easy Serum/Plasma Detection Kit (Qiagen, USA) according to the manufacturer's protocol. RT-PCR analysis was carried out as described elsewhere.[8] RNA was reverse transcribed to cDNA with gene-specific primers. The relative expression was assessed using the equation 2−dCT, where dCT= (CmiRNA− CRNU). Unless otherwise mentioned Student's t -test (2 tails, type 2) was used. P ≤0.05 was considered statistically significant.


 > Results Top


Blood samples obtained from histopathologically proven cases of lung AC and SCC were used for molecular diagnosis. We evaluated the expression of six novel miRNAs which are not reported before in blood sample of lung cancer cases.

MicroRNA expression in lung adenocarcinoma

Plasma was separated from peripheral blood of AC patients and used for the estimation of miRNA expression [Supplementary Figure 1]. miRNAs mir-2114 ( P < 0.001), mir-449c ( P < 0.001), and mir-2116 ( P < 0.01) were found to be significantly present in AC cases as compared to control. mir-2114 and mir-449c showed trend for high expression compared to mir-2116 [Figure 1].
Figure 1: MicroRNA expression in plasma of adenocarcinoma patient. Total RNA was extracted from plasma separated from peripheral blood of adenocarcinoma patient. The expression was quantitated for mir-2114, mir-2115, mir-2116, mir-2117, mir-449c, and mir-548q after normalizing it with loading control Caenorhabditis elegans RNA. Data shown as mean ± standard deviation; n = 30 adenocarcinoma patients and 20 controls, *** P < 0.001, ** P < 0.01 compared with control

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MicroRNA expression in lung squamous cell carcinoma

In SCC, mir-2115, mir-2114, mir-548q, and mir-2117 were upregulated as compared to control. Among these miRNAs, mir-2115 showed the highest expression, whereas the overall expression of mir-2114, mir-548q, and mir-2117 was similar [Figure 2].{Figure 2}

MicroRNA expression in lung adenocarcinoma cell line

The total RNA was extracted from exponentially growing lung AC cell line (A549) and human embryonic kidney cells (HEK293T) as a control. The expression was normalized by internal control RNU_62_4.

In A549 cells, the expression of mir-2114, mir-2116, mir-449c, and mir-549q was significantly upregulated, whereas the level of mir-2115 and mir-2117 was not significantly different as compared to control [Figure 3].
Figure 3: Expression of microRNAs in A549 and 293T cells. Total RNA was extracted from exponentially growing A549 and 293T cells, and cDNA was synthesized. Expression of mir-2114, mir-2115, mir-2116, mir-2117, mir-449c, and mir-548q was evaluated after normalizing the samples by internal control RNU 62_4. Fold expression of different microRNA in A549 over 393T cells was estimated

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Differential expression of microRNAs in adenocarcinoma and squamous cell carcinoma

Through this molecular biomarker-based screening, we found that all the miRNAs showed different levels of expression in AC and SCC. Mir-2115, mir-548q, and mir-2117 were found to be expressed only in SCC cases, whereas mir-2116 and mir-449c were detected exclusively in AC. Mir-2114 was found to be expressed in both AC and SCC, although the expression was higher in AC [Table 1].
Table 1: Differential expression of microRNAs in peripheral blood of adenocarcinoma and squamous cell carcinoma

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Among all the miRNAs evaluated in lung AC and SCC, the expression of mir-2115 and mir-449c was highest in SCC and AC, respectively. This implies that these miRNAs are potential biomarkers for the diagnosis of these two subtypes of non-small cell lung carcinoma.


 > Discussion Top


An early detection of cancer has been an incentive for clinicians. With current diagnostic modalities such as MRI, computerized tomography, ultrasonography, fine-needle aspiration cytology, and CT-guided biopsy, the diagnosis is usually not possible during early stages of the disease process.[9],[10] Over the last decade, research is focused on the identification of reliable biomarkers to reduce tumor-associated mortality. Molecular-based methods such as identification of miRNAs are promising biomarkers and carry significant potential in translational medicine.[11] Previous studies have demonstrated that miRNAs in circulation are present in exosomes and are, therefore, highly stable against RNase activity.[12] Changes in the levels of miRNAs in circulation reflect the alterations at the tissue level.

miRNAs evaluated in lung cancer have been previously characterized for their tumorigenic role in other malignancies. Mir-548q is a known oncogene and prognostic marker in gastric cancer.[13] In this study, through the identification of novel miRNAs in peripheral blood of AC and SCC, we have established a method to differentially diagnose nonsmall-lung carcinoma subtypes. These results can be similarly applied to other lung cancer subtypes for novel marker identification.

The merits of this study include the involvement of two different subtypes of lung cancer cases and validation of data in lung AC cell line. However, this study has some potential limitations too. First, the sample size in individual cancer subtype was small. Second, we could not verify the data in lung SCC cell line.

In summary, the current study has significant diagnostic prospects. We have shown that an early and accurate diagnosis of lung carcinoma is possible with the evaluation of circulating miRNAs. This miRNA-based differential diagnosis can also be combined with other diagnostic modalities for precise patient management. The method is suitable for screening a large cohort of high- and low-risk individuals for cancer interception. Further, sample collection in such cases is minimally invasive and can be carried out during routine follow-up of cases for monitoring the effectiveness of treatment.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394-424.  Back to cited text no. 1
    
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Sutedja G. New techniques for early detection of lung cancer. Eur Respir J Suppl 2003;39:57s-66s.  Back to cited text no. 2
    
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Hirsch FR, Franklin WA, Gazdar AF, Bunn PA Jr. Early detection of lung cancer: Clinical perspectives of recent advances in biology and radiology. Clin Cancer Res 2001;7:5-22.  Back to cited text no. 3
    
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Sokolenko AP, Imyanitov EN. Molecular diagnostics in clinical oncology. Front Mol Biosci 2018;5:76.  Back to cited text no. 4
    
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Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, et al . Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 2008;105:10513-8.  Back to cited text no. 5
    
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Adachi T, Nakanishi M, Otsuka Y, Nishimura K, Hirokawa G, Goto Y, et al . Plasma microRNA 499 as a biomarker of acute myocardial infarction. Clin Chem 2010;56:1183-5.  Back to cited text no. 6
    
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Weber JA, Baxter DH, Zhang S, Huang DY, Huang KH, Lee MJ, et al . The microRNA spectrum in 12 body fluids. Clin Chem 2010;56:1733-41.  Back to cited text no. 7
    
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Yuan JS, Reed A, Chen F, Stewart CN Jr., Statistical analysis of real-time PCR data. BMC Bioinformatics 2006;7:85.  Back to cited text no. 8
    
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Chojniak R, Pinto PN, Ting CJ, Cohen MP, Guimarães MD, Yu LS, et al . Computed tomography-guided transthoracic needle biopsy of pulmonary nodules. Radiol Bras 2011;44:315-20.  Back to cited text no. 9
    
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Chojniak R, Grigio HR, Bitencourt AG, Pinto PN, Tyng CJ, Cunha IW, et al . Percutaneous computed tomography-guided core needle biopsy of soft tissue tumors: Results and correlation with surgical specimen analysis. Radiol Bras 2012;45:259-62.  Back to cited text no. 10
    
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Hussein AA, Forouzanfar T, Bloemena E, de Visscher J, Brakenhoff RH, Leemans CR, et al . A review of the most promising biomarkers for early diagnosis and prognosis prediction of tongue squamous cell carcinoma. Br J Cancer 2018;119:724-36.  Back to cited text no. 11
    
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Simons M, Raposo G. Exosomes –Vesicular carriers for intercellular communication. Curr Opin Cell Biol 2009;21:575-81.  Back to cited text no. 12
    
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Liu J, Ni X. MicroRNA-548q is a novel oncogene and potential prognostic biomarker in human gastric cancer. Int J Clin Exp Pathol 2017;10:2689-700.  Back to cited text no. 13
    


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